Two vampires known as Death Dealers track a pair of Lycans, an ancient species of werewolf, who are in their human form. The vampires, Selene, (Kate Beckinsale) and Rigel, specialize in assassinating Lycans. Selene's motivation goes beyond duty; she also wants revenge; Lycans slaughtered her family when she was a child. The vampires believe they defeated the werewolves many centuries ago and killed their leader, Lucian, and that they must kill off the survivors. As the vampires follow the Lycans into a subway station the werewolves open fire with submachine guns and Rigel is killed. After the Lycans retreat from Selene's return fire and the arrival of a third Death Dealer, Nathaniel, she tracks them to their den. Nathaniel is slain by a Lycan named Raze after being ambushed. Upon reaching the Lycan den, Selene hears loud howling and learns that the Lycans have developed a new high-tech bullet to kill the vampires.
When Selene arrives at the vampire coven's mansion she recounts the evening's events and urges an attack on the Lycans, but the vampire regent, Kraven, tells her to drop the matter. Selene determines that the Lycans may have been following a human, Michael Corvin, and continues her investigation. In an underground Lycan lair, a scientist named Singe tests blood from kidnapped descendants of the ancient Corvinus family, trying to find a pure source of the ancient and powerful Corvinus bloodline. Soon after Selene finds Michael the pair are attacked by six lycans, including Lucian. After Lucian bites Michael, Selene helps Michael escape and the two become attracted to each other.
Selene discovers that when Lucian was supposedly killed, Kraven was the only witness. Sensing a conspiracy, she wakes a powerful elder vampire, Viktor, who has been hibernating. Viktor is angered by his early awakening, as it was Markus' turn to rule, not his. Viktor believes Kraven and orders Selene to acquiesce to the vampire hierarchy. Kraven has secretly planned to kill the vampire elder, Amelia, and her companions, with Lucian's help.
Selene tells Michael about the feud and her past. She then chains him to a wall, fearing that when he changes at the full moon he will kill innocent people. Selene captures Singe while Michael is captured by two Lycans. Selene returns to the mansion with Singe, who admits to Viktor that the Lycans are trying to combine the bloodlines of the two species with the Corvinus Strain to create a powerful hybrid. After Kraven flees the mansion because Singe has revealed that Kraven and Lucian are working together, news arrives that Amelia, who was coming to awaken Markus, has been killed by Lycans. Viktor kills Singe and gives Selene the chance to redeem herself by killing Michael.